U.S. patent application number 10/400024 was filed with the patent office on 2004-01-08 for autofluorescence detection and imaging of bladder cancer realized through a cystoscope.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Demos, Stavros G., deVere White, Ralph W..
Application Number | 20040006276 10/400024 |
Document ID | / |
Family ID | 46299083 |
Filed Date | 2004-01-08 |
United States Patent
Application |
20040006276 |
Kind Code |
A1 |
Demos, Stavros G. ; et
al. |
January 8, 2004 |
Autofluorescence detection and imaging of bladder cancer realized
through a cystoscope
Abstract
Near infrared imaging using elastic light scattering and tissue
autofluorescence and utilizing interior examination techniques and
equipment are explored for medical applications. The approach
involves imaging using cross-polarized elastic light scattering
and/or tissue autofluorescence in the Near Infra-Red (NIR) coupled
with image processing and inter-image operations to differentiate
human tissue components.
Inventors: |
Demos, Stavros G.;
(Livermore, CA) ; deVere White, Ralph W.;
(Sacramento, CA) |
Correspondence
Address: |
Michael C. Staggs
Assistant Laboratory Counsel
Lawrence Livermore National Laboratory
P.O. Box 808, L-703
Livermore
CA
94551
US
|
Assignee: |
The Regents of the University of
California
|
Family ID: |
46299083 |
Appl. No.: |
10/400024 |
Filed: |
March 25, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10400024 |
Mar 25, 2003 |
|
|
|
10190231 |
Jul 5, 2002 |
|
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Current U.S.
Class: |
600/476 |
Current CPC
Class: |
A61B 1/0669 20130101;
A61B 1/313 20130101; A61B 5/0075 20130101; A61B 1/043 20130101;
G01N 21/49 20130101; A61B 1/063 20130101; A61B 5/0086 20130101;
A61B 1/0638 20130101; A61B 1/0646 20130101; A61B 5/0071 20130101;
A61B 1/307 20130101; A61B 1/00186 20130101; G01N 21/6445
20130101 |
Class at
Publication: |
600/476 |
International
Class: |
A61B 006/00 |
Goverment Interests
[0002] The United States Government has rights in this invention
pursuant to Contract No. W-7405-ENG-48 between the United States
Department of Energy and the University of California for the
operation of Lawrence Livermore National Laboratory.
Claims
The invention claimed is:
1. A diagnostic apparatus, comprising: at least one electromagnetic
radiation source directed to illuminate one or more tissue
components, an interior examination device adapted to transmit the
radiation source for illumination of the tissue components and
further adapted to relay a near-infrared scattered and/or an
autofluorescence emission radiation of the illuminated tissue
components, a detector adapted to capture from the tissue
components, a near-infrared scattered and/or a near infrared
autofluorescence emission radiation that is relayed by the interior
examination device; and means to characterize the captured
near-infrared scattered and/or near infrared autofluorescence
emission radiation from the tissue components.
2. The apparatus of claim 1, wherein the detector includes an
on-chip charge amplification CCD camera.
3. The apparatus of claim 1, wherein the interior examination
device includes a device selected from cystoscopes, ureterscopes,
and endoscopes.
4. The apparatus of claim 3, wherein the interior examination
device includes a non-flexible cystoscope capable of transurethral
resection.
5. The apparatus of claim 3, wherein the interior examination
device includes a flexible cystoscope.
6. The apparatus of claim 3, wherein the interior examination
device includes a cystoscope adapted to relay an image to the
detector with an operatively coupled image preserving optical fiber
bundle.
7. The apparatus of claim 1, wherein the electromagnetic radiation
source is a broadband lamp arranged to illuminate the tissue
components with one or more predetermined substantially narrow band
filtered wavelengths of at least 400 nm.
8. The apparatus of claim 1, wherein the electromagnetic radiation
source includes one or more substantially narrow band laser sources
arranged to illuminate the tissue components with a respective
wavelength of at least 400 nm.
9. The apparatus of claim 1, wherein a second illumination assembly
directs at least two predetermined substantially narrow band
wavelengths to illuminate the tissue components.
10. The apparatus of claim 1, wherein the characterization means
includes a computer and image processing software and/or human
interpretation of displayed real time images of tissue component
features that are important to a trained operator.
11. The apparatus of claim 10, wherein one or more inter-image
operations are performed by the image processing software.
12. The apparatus of claim 11, wherein the inter-image operations
includes software operations on a detected scattered radiation from
the laser sources and/or polarization states from the tissue
components to enhance image contrast and visibility of the tissue
components under examination.
13. The apparatus of claim 11, wherein the inter-image operations
are between at least two images selected from a near-infrared
cross-polarized light scattering image of a first wavelength, a
near-infrared cross-polarized light scattering image of a second
wavelength, a near-infrared parallel-polarized light scattering
image of a first wavelength, a near-infrared parallel-polarized
light scattering image of a second wavelength, a near-infra-red
autofluorescence image under a first excitation wavelength, a
near-infra-red autofluorescence image under a second excitation
wavelength, an orthogonal-polarization component of a near-infrared
autofluorescence image produced by a polarized excitation, and a
parallel-polarization component of the near-infrared
autofluorescence image produced by a polarized excitation to
enhance image contrast and visibility of one or more tissue
components.
14. The apparatus of claim 1, wherein the detector captures an
analyzed degree of polarization near-infrared scattered
electromagnetic radiation and/or near infrared autofluorescence
emission.
15. The apparatus of claim 14, wherein the laser sources are
polarized and wherein the analyzed degree of polarization comprises
one or more polarizers adapted to analyze the scattered
electromagnetic radiation and the near infra-red autofluorescence
emission received from the tissue components by the detector.
16. The apparatus of claim 9, wherein the detector includes a
two-dimensional CCD such that the spectral image information
produced by the illuminated tissue components are capable of being
simultaneously projected on different parts of the detector.
17. The apparatus of claim 1, wherein the apparatus is capable of
imaging and differentiating one or more grades of bladder
tumors.
18. The apparatus of claim 1, wherein the apparatus includes six
laser sources.
19. A diagnostic apparatus, comprising: at least two substantially
narrow-band electromagnetic radiation wavelength sources directed
to illuminate one or more tissue components, an interior
examination device adapted to transmit the wavelength sources for
illumination of the tissue components and further adapted to relay
a near-infrared scattered radiation of the illuminated tissue
components, a detector adapted to simultaneously capture one or
more images produced by the near-infrared scattered radiation from
the tissue components; and means to characterize the captured
near-infrared scattered radiation from the tissue components.
20. The apparatus of claim 19, wherein the detector includes a
two-dimensional CCD such that the spectral information produced by
the illumination of tissue components by the wavelength sources are
capable of being simultaneously projected on different parts of the
detector.
21. The apparatus of claim 19, wherein the interior examination
device includes a non-flexible cystoscope capable of transurethral
resection.
22. The apparatus of claim 19, wherein the interior examination
device includes a flexible cystoscope.
23. The apparatus of claim 19, wherein the interior examination
device includes a cystoscope adapted to relay an image to the
detector with an operatively coupled image preserving optical fiber
bundle.
24. The apparatus of claim 19, wherein the characterization means
includes a computer and image processing software and/or human
interpretation of displayed real time images of tissue component
features that are important to a trained operator.
25. The apparatus of claim 24, wherein one or more inter-image
operations are performed by the image processing software on the
images.
26. The apparatus of claim 25, wherein the inter-image operations
include operations on a detected scattered radiation from the
source wavelengths and/or polarization states from the tissue
components to enhance image contrast and visibility of the tissue
components under examination.
27. The apparatus of claim 25, wherein the inter-image operations
are between at least two images selected from a near-infrared
cross-polarized light scattering image of a first wavelength, a
near-infrared cross-polarized light scattering image of a second
wavelength, a near-infrared parallel-polarized light scattering
image of a first wavelength, a near-infrared parallel-polarized
light scattering image of a second wavelength produced by a
polarized excitation to enhance image contrast and visibility of
one or more tissue components.
28. The apparatus of claim 19, wherein the detector captures an
analyzed degree of polarization near-infrared scattered
electromagnetic radiation.
29. The apparatus of claim 28, wherein the degree of polarization
comprises one or more optical polarizers adapted to polarize the
wavelength sources and wherein the analyzed degree of polarization
comprises one or more polarizers adapted to analyze the scattered
electromagnetic radiation received from the tissue components by
the detector.
30. The apparatus of claim 19, wherein the apparatus is capable of
imaging and differentiating one or more grades of bladder
tumors.
31. A diagnostic apparatus, comprising: one or more laser sources
directed to illuminate one or more tissue components, a cystoscope
adapted to transmit the laser sources for illumination of the
tissue components and further adapted to relay a near-infrared
scattered electromagnetic and/or a near infrared autofluorescence
emission radiation of the illuminated tissue components, an on-chip
charge amplification CCD adapted to capture images produced by a
near-infrared scattered electromagnetic and/or a near infrared
autofluorescence emission radiation from the tissue components; and
a computer configured with an image processing software to
characterize the captured near-infrared scattered and/or
near-infrared autofluorescence emission radiation from the tissue
components.
32. A diagnostic apparatus, comprising: at least two laser sources
directed to illuminate one or more tissue components, a cystoscope
adapted to transmit the laser sources for illumination of the
tissue components and further adapted to relay a scattered
near-infrared radiation produced by the illuminated tissue
components, a two-dimensional CCD, adapted to simultaneously
capture images produced by the scattered near-infrared radiation
from the tissue components; and a computer configured with an image
processing software to characterize the captured near-infrared
scattered radiation from the tissue components.
33. A diagnostic method, comprising: interrogating one or more
tissue components with an interior examination device, wherein the
device is capable of directing one or more substantially narrow
band electromagnetic radiation sources to provide illumination of
one or more tissue components and further capable of relaying a
near infrared scattered and/or a near infrared autofluorescence
emission radiation from the illuminated tissue components,
detecting the near infrared scattered and/or the near infrared
autofluorescence emission radiation from the tissue components; and
characterizing the detected near-infrared scattered and
autofluorescence emission radiation from the tissue components.
34. The method of claim 33, wherein the detecting step includes an
on-chip charge amplification CCD camera.
35. The method of claim 33, wherein the detecting step includes a
two-dimensional CCD such that the spectral image components
produced by the laser sources are capable of being simultaneously
projected on different parts the detector.
36. The method of claim 33, wherein the interior examination device
includes a non-flexible cystoscope capable of transurethral
resection.
37. The method of claim 33, wherein the interior examination device
includes a flexible cystoscope.
38. The method of claim 33, wherein the interior examination device
is a cystoscope adapted to relay an image with an operatively
coupled image preserving optical fiber bundle.
Description
RELATED APPLICATION
[0001] This application is a Continuation-In-Part of application
Ser. No. 10/190,231 filed Jul. 5, 2002, and claims priority
thereto.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to a medical diagnostic for
the examination of tissue components. Specifically, the present
invention relates to an optical imaging method and apparatus for
in-vivo and real-time imaging of bladder cancer and determination
of tumor margins.
[0005] 2. Description of Related Art
[0006] Diagnostic medical equipment typically includes
time-consuming, bulky, expensive apparatus that often exposes human
tissue components to potentially harmful radiation and or
chemicals. Optical methods and systems for the identification of
objects that possess different optical properties or abnormal
compositions embedded in scattering media such as, human tissue,
are desirable because such systems can be designed as compact,
inexpensive, portable, and non-invasive spectral investigative
tools. Optical spectroscopy, as one such optical method example,
has been widely used to acquire fundamental knowledge about
physical, chemical, and biological processes that occur in
biomaterials. Most tissue spectroscopy research has employed UV to
visible light sources from 250-nm to 600-nm. The main active
fluorophores in this spectral region are tryptophan, collagen,
elastin, NAD(P)H, flavins and prophyrins. The disadvantage of these
wavelengths is their short photon penetration depth in tissues,
which leads to extraction of information only from superficial
tissue layers.
[0007] Accordingly, a need exists for optical diagnostic methods
and systems to be utilized in a compact portable system to recover
optical information with regard to human tissue and organ
compositions.
SUMMARY OF THE INVENTION
[0008] Accordingly, the present invention provides a diagnostic
apparatus that includes: at least one electromagnetic radiation
source, an arranged interior examination device adapted to transmit
the radiation source and further adapted to relay a near-infrared
scattered and/or an autofluorescence emission radiation, and a
detector adapted to capture the scattered and/or near-infrared
emission radiation produced by the radiation sources and optically
coupled to the interior examination device so that characterization
of the one or more tissue components is capable of being
performed.
[0009] Another aspect of the present invention provides a
diagnostic apparatus that includes: at least two substantially
narrow-band electromagnetic radiation wavelength sources for
simultaneous illumination of one or more tissue components, an
arranged interior examination device adapted to transmit the
wavelength sources for illumination of the tissue components and
further adapted to relay a near-infrared scattered radiation of the
illuminated tissue components; and a detector adapted to
simultaneously capture one or more images produced by the
near-infrared scattered radiation from the tissue components so
that characterization of the one or more tissue components is
capable of being performed.
[0010] Another aspect of the present invention provides a
diagnostic apparatus that includes: one or more laser sources, a
cystoscope adapted to transmit laser source emission and further
adapted to relay a near-infrared scattered electromagnetic and/or a
near infrared autofluorescence emission radiation; and an on-chip
charge CCD adapted to capture the scattered and/or near-infrared
emission produced by the laser sources, wherein a computer
configured with an image processing software can characterize the
captured near-infrared scattered and/or near-infrared
autofluorescence emission radiation from the tissue components.
[0011] Another aspect of the present invention provides a
diagnostic apparatus that includes: at least two laser sources for
simultaneous illumination of one or more tissue components, a
cystoscope adapted to transmit laser source illumination and
further adapted to relay near-infrared scattered radiation; and a
two-dimensional CCD adapted to simultaneously capture the scattered
emission produced by the laser sources so that a computer
configured with an image processing software can characterize the
captured near-infrared scattered radiation from the tissue
components.
[0012] Still another aspect of the present invention is directed to
an improved diagnostic imaging method for medical applications
comprising the steps of: interrogating one or more tissue
components with an interior examination device capable of directing
one or more substantially narrow band electromagnetic radiation
sources to provide illumination of one or more tissue components
and further capable of relaying a near infrared scattered and/or a
near infrared autofluorescence emission radiation from the
illuminated tissue components, detecting a scattered
electromagnetic radiation and/or a near infrared autofluorescence
emission from the tissue components; and characterizing the
detected scattered electromagnetic radiation and/or
autofluorescence emission from said tissue components.
[0013] Accordingly, the present tissue imaging system and method
provides a portable, cost effective, non-invasive arrangement,
capable of directing substantially monochromatic electromagnetic
radiation and capable of providing spectrally processed images in
the much desired need for differentiating components in human
and/or animal tissues.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The accompanying drawings, which are incorporated into and
constitute a part of the specification, illustrate specific
embodiments of the invention and, together with the general
description of the invention given above, and the detailed
description of the specific embodiments, serve to explain the
principles of the invention.
[0015] FIG. 1 is a simplified diagram of an exemplary medical
tissue imaging apparatus incorporating elastic light scattering,
fluorescence imaging, and image processing capabilities.
[0016] FIG. 2 shows a series of breast tissue images collected by
the present invention.
[0017] FIG. 3 shows a series of malignant and benign tumor images
from a liver specimen collected by the present invention.
[0018] FIG. 4 shows a series of uterine images collected by the
present invention.
[0019] FIG. 5 shows a series of bladder images collected by the
present invention.
[0020] FIG. 6(a) shows a schematic layout of the main components of
an example cystoscopic diagnostic system.
[0021] FIG. 6(b) shows a cross-sectional view of the output tip of
an example cystoscope.
DETAILED DESCRIPTION OF THE INVENTION
[0022] Referring now to the drawings, specific embodiments of the
invention are shown. The detailed description of the specific
embodiments, together with the general description of the
invention, serves to explain the principles of the invention.
[0023] Unless otherwise indicated, all numbers expressing
quantities of ingredients, constituents, reaction conditions and so
forth used in the specification and claims are to be understood as
being modified in all instances by the term "about." Accordingly,
unless indicated to the contrary, the numerical parameters set
forth in the specification and attached claims are approximations
that may vary depending upon the desired properties sought to be
obtained by the subject matter presented herein. At the very least,
and not as an attempt to limit the application of the doctrine of
equivalents to the scope of the claims, each numerical parameter
should at least be construed in light of the number of reported
significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting
forth the broad scope of the subject matter presented herein are
approximations, the numerical values set forth in the specific
examples are reported as precisely as possible. Any numerical
value, however, inherently contain certain errors necessarily
resulting from the standard deviation found in their respective
testing measurements.
[0024] General Description
[0025] The present invention combines monochromatic laser sources,
a broadband light source, optical filtering, a computer, optical
imaging, and computer software capable of image analysis that
includes inter-image operations. A useful feature of the present
invention is that fresh surgical resections collected from patients
may be measured in-vitro (e.g., in an artificial environment) and
in-vivo (e.g., during medical biopsy or intervention procedures)
immediately upon collection. In addition, the system has particular
utility as a tissue component interrogation tool for human tissue
specimens, such as, but not limited to, kidney, uterine, bladder,
breast, liver, adipose, abnormal (i.e., contrary to normal
structure), normal, and veins and/or nerves from surrounding muscle
tissue.
[0026] Accordingly, the present invention provides a non-invasive
optical analysis means suitable for rapid, in-vitro or in-vivo
differentiation of human tissue components.
[0027] Specific Description
[0028] Turning now to the drawings, a diagram that illustrates
exemplary embodiments of a system constructed in accordance with
the present invention is shown in FIG. 1. The system, designated
generally by the reference numeral 100, provides a method and
apparatus for interrogating and characterizing human tissue
components in a clinical environment from a specimen. System 100,
as shown in FIG. 1, designed as a portable, compact apparatus,
includes the following basic components: a CPU with software for
sample image processing (not shown); a lens system 10 for image
collection; an image acquisition system 12; a substantially
monochromatic 20 light source; an optical band-pass filters 62; an
optical polarization filters 58; a sample holder (not shown); a
white light source 80; and the remaining components (discussed
below).
[0029] The sample image processing software (not shown) can be a
commercially available product. However, any image analysis
software capable of performing image processing with inter-image
operations may be employed with the present invention to provide
tissue component differentiation.
[0030] Lens system 10, for example a camera lens, is coupled to
image acquisition system 12, such as, but not limited to, a liquid
nitrogen cooled CCD camera, a two-dimensional array detector, an
avalanche CCD photodetector, a photomultiplier or a photodiode
capable of point by point scanning. However, any imaging device
constructed to the design output parameters for image acquisition
system 12 can also be employed in the present invention. Such
exemplary image acquisition systems 12 capable of performing
time-integrated images may be employed with the present invention
to image illuminated tissue sample 46 housed in a kinematically
(i.e., can be removed and rapidly repositioned with accuracy)
mounted sample holder (not shown).
[0031] Monochromatic light sources 20 is a low power laser having
an output power of at least 1 mW that operates at 632 nm. However,
any laser source capable of providing a wavelength and intensity
capable of differentiating tissue components may be employed with
the present invention. White light source 80 provides a broadband
of wavelengths for the scattering experiments. White light source
80 and monochromatic light source 20 are brought into the imaging
compartment (not shown) by an optical means such as, a plurality of
large core optical fibers 52, and 88 (i.e., multimode fibers).
[0032] Monochromatic light source 20 is used for photoexcitation to
provide NIR autofluorescence images, including a polarizer 58 and a
narrow band-pass filter 62 positioned to ensure a predetermined
narrow band of electromagnetic radiation with a predetermined
polarization orientation to uniformly illuminate sample 46. White
light source 80, to provide elastic light scattered images,
additionally has one or more polarization filters 90 positioned to
orient one or more illumination output beams 92 to a predetermined
polarization orientation prior to uniformly illuminating a tissue
specimen.
[0033] Sample 46 is illuminated with a set of one or more images,
preferably seven, recorded for each sample. The objective is to
employ hyperspectral (i.e., using various spectroscopic techniques
and multiple wavelength and/or spectral bands) imaging to
investigate the ability of polarized light in combination with
native NIR tissue autofluorescence to image and differentiate human
tissue components. An exemplary useful benefit of the present
invention is to image and differentiate human tissue components,
such as, but not limited to, cancerous growth from contiguous
normal tissue or nerves and/or vascular structures from muscle.
[0034] The combined investigative approach method embodiment of
polarized light scattering with NIR native tissue autofluorescence
under long-wavelength laser excitation to reveal optical
fingerprint characteristics for tissue components can be initiated
by either of the following two methods of the present
invention.
[0035] Autofluorescence
[0036] A related method for using autofluorescence emission (i.e.,
the spectral wing emission greater than 600 nm) to detect cancerous
tissue is disclosed in Incorporated by reference Co-pending,
Co-filed U.S. application Ser. No. xxx,xxx titled "Method And
Apparatus For Examining A Tissue Using The Spectral Wing Emission
therefrom Induced By Visible To Infrared Photoexcitation" by Alfano
et al., the disclosure is herein incorporated by reference in its
entirety. NIR autofluorescence, as disclosed in the above
referenced co-pending U.S. application, is particularly useful for
monitoring and/or imaging the porphyrin content in tissues.
Porphyrins, effective at transferring electrons in sub-cellular
organelles known as mitochondria are known to fluoresce in the
visible light portion of the luminescence spectra. In addition,
there is strong evidence that the heme-biosynthetic pathway, and
therefore the production of porphyrins is disturbed in any new and
abnormal growth, such as, cancer tissue. Thus, NIR autofluorescence
of fluorophores, such as, but not limited to, porphyrins, provides
one such exemplary medical diagnostic means of enhancing the
contrast between normal and cancerous tissue components. However,
the present invention provides a useful improvement thereof by
incorporating autofluorescence images, image processing coupled
with inter-image manipulations with elastic light scattering images
discussed below to produce high contrast, high visibility images
capable of differentiating substantially various human tissue
components from a specimen, such as, but not limited to, normal and
tumor tissue.
[0037] Turning again to FIG. 1, system 100 includes monochromatic
light source 20 that transmits laser light through optical fiber
52. As one embodiment, monochromatic light source 20 may include a
Helium-neon laser that operates at about 633 nm. However, any
monochromatic laser light source operating at wavelengths from
about 250 nm to about 1100 nm can be employed to provide NIR
autofluorescence emission images for the present invention. Light
source 20 is selected by a remote control pad (not shown) and
directed by optical fiber 52 into an imaging compartment (not
shown). Narrow-band filter 58 having an optical band-pass of at
least 10 nm, and polarizer, 62 capable of producing linear,
circular or elliptical polarization are positioned at the distal
end of optical fiber 52 to ensure a degree of polarized
monochromatic light beam 66 received from light source 20. The
output of optical fiber 52 is constructed to provide beam 66 with a
diverging property to substantially uniformly illuminate sample
46.
[0038] FIG. 1 also shows an alternate embodiment that includes
monochromatic light source 30, such as, an Nd:YAG diode-pumped
solid-state laser that operates at 532 nm, to transmit laser light
through optical fiber 54. Light source 30 is selected by the remote
control pad (not shown) and directed by optical fiber 54 into the
imaging compartment (not shown). Narrow-band filter 60 having an
optical band-pass of at least 10 nm, and polarizer, 64 capable of
producing linear, circular or elliptical polarization are
positioned at the distal end of optical fiber 54 to ensure a degree
of polarized monochromatic light beam 68 received from light source
30. The output of optical fiber 54 is constructed to provide beam
68 with a diverging property to substantially uniformly illuminate
sample 46.
[0039] Autofluorescence emission is generated from light sources
20, or 30, and then collected from tissue sample 46 in a
back-scattering geometry, as generally shown by optical rays 94, by
lens system 10 having one or more interchangeable camera lenses,
preferably a 50-mm focal length camera lens. An analyzing polarizer
74 is positioned before lens system 10 on a translation stage such
that parallel linear polarization, orthogonal cross-polarization,
orthogonal elliptical polarization, same elliptical polarization,
opposite circular polarization, or non-polarization analysis of the
autofluorescence emission may be employed. A bandpass filter 72 is
additionally positioned before lens system 10 to ensure a proper
spectral band selection between about 650 nm and about 1500 nm for
imaging. The autofluorescence emission is time gated for 0.1 or
more seconds and recorded by image acquisition system 12,
preferably a Princeton Instrument Model No. TE/CCD-512 liquid
nitrogen cooled camera. The output of image acquisition system 12
is coupled to a computer (not shown), e.g., a laptop computer, and
image processed by commercially available image processing
software, such as, Roper Scientific Winspec/32 and/or Winview/32
software, and displayed on, for example, a computer screen for
human eye diagnosis or for image software analysis.
[0040] Elastic Light Scattering
[0041] The NIR polarized elastic light scattering method of the
present invention to delineate differences in absorption and
scattering in human tissue components allows an end-user to acquire
clinical diagnostic deep-subsurface (e.g., at least 1 cm) images.
An illumination wavelength, preferably greater than 500 nm from an
electromagnetic radiation source is utilized to provide mean photon
penetration depth larger than 1 mm. Linear cross-polarization and
spectral analysis of the scattered photons substantially removes
the photon information from the orthogonal illumination
polarization resulting from the surface and allows substantially
all of the scattered photons from the subsurface tissue to be
imaged.
[0042] In addition, a spectral polarization difference technique
(SPDI) and NIR illumination related method embodiment is utilized
in the present invention and is disclosed in incorporated by
reference, U.S. application Ser. No. 5,847,394, titled "Imaging of
objects based upon the polarization or depolarization of light," by
Alfano et al., the disclosure which is herein incorporated by
reference in its entirety. With SPDI, different illumination
wavelengths are utilized to record images having a differential
mean photon penetration depth. Thus, a smaller differential in the
illumination wavelengths provides narrower differential depth zones
while a larger difference in two exemplary illuminating wavelengths
gives rise to a wider depth zone. Cross-polarization and
normalization analysis coupled with inter-image operations, such
as, but not limited to, subtraction between one or more
illuminating wavelengths provide information as to the tissue
structure between the penetration depths of the one or more
respective probe illumination wavelengths. However, the present
invention provides a useful improvement thereof, as similarly
discussed herein before, by incorporating inter-image operations of
autofluorescence images and light scattering, such as, single
wavelength cross-polarized light scattered images, to provide
higher visibility and contrast information from images for humans
tissue component differentiation.
[0043] Accordingly, the present invention utilizes NIR
autofluorescence, NIR light scattering, inter-image operations
between individual methods, (preferably inter-image operations
incorporating both autofluorescence and light scattering), to
provide differential tissue component information within one or
more exemplary images.
[0044] Turning again to FIG. 1, a broad-band, i.e., its
electromagnetic spectrum covers the visible, (e.g., 0.4 .mu.m to
0.7 .mu.m), and a substantial amount of the invisible, (e.g., 0.7
.mu.m to 2.0 .mu.m), white light source 80 is selected by the
remote pad (not shown) to transmit up to 100 watts of white light
to coupled optical fiber bundle 82. Fiber bundle 82 has its distal
end coupled to a tunable filter (not shown) or a filter wheel 84
that is remotely controlled by a filter wheel remote pad (not
shown) for insertion of a predetermined optical filter (not shown).
Such a predetermined narrow-band (e.g., 40 nm) interference filter
in the visible or invisible between about 700-nm and about 1000-nm
spectral range can thus be rapidly positioned at the output distal
end of fiber bundle 82. However, any type of band-pass filter
means, for example broad spectral band filters or long-pass
interference filters can be employed in practice of the
invention.
[0045] A manifold 86 is coupled to an optical delivery means, such
as, one or more large core optical fibers 88, preferably four.
Manifold 86 is additionally coupled to filter wheel 84 to receive
and direct the selected wavelength band through optical fibers 88
in order to substantially uniformly illuminate sample 46. One or
more linear polarizers 90 are constructed at the output of one or
more optical fibers 88 to ensure a degree of linear polarization of
one or more scattering diverging illumination output beams 92
having predetermined selected wavelength bands.
[0046] The elastically scattered light is then collected from
tissue sample 46 in a back-scattering geometry, as generally shown
by optical rays 94, by lens system 10 similar to the
autofluorescence measurements discussed above. Analyzing polarizer
74 is positioned on a translation stage such that parallel
polarization, cross-polarization or non-polarization analysis of
the elastic light scattering may be employed. Image acquisition
system 12 is time gated for 0.1 or more seconds and one or more
images as a result of the elastic light scattering from the sample
is recorded. The output of image acquisition system 12 is coupled
to a computer (not shown), e.g., a laptop computer, image
processed, and displayed on, for example, one or more computer
screens for human eye diagnosis or for image software analysis as
previously discussed.
[0047] An exemplary prototype was constructed and positioned in a
lab space located at the UC Davis Medical Center in Sacramento,
Calif. The following autofluorescence emission and elastic light
scattering images illustrating adipose, cancerous and contiguous
normal human tissue, obtained from fresh surgical resections from
more than 60 patients are used to only illustrate some of the novel
imaging capabilities of the present invention.
[0048] FIG. 2 illustrates a capability of the present invention
with a set of images of an approximately 4-cm.times.3-cm human
breast tissue specimen 204 with multifocal high grade ductal
carcinoma 210 shown in FIG. 2d, surrounded by fibrous supporting
tissue 215 with an adjacent area of fatty 220 (i.e., adipose)
infiltration as shown in FIG. 2f. FIGS. 2a and 2b show
autofluorescence images in the 700-nm and 1000-nm spectral region
under a 532-nm and a 633-nm substantially monochromatic
illumination, respectively. FIG. 2c shows a light scattering image
of specimen 204 under 700-nm illumination. FIG. 2d shows a novel
feature of the present invention wherein a ratio of the
autofluorescence image of FIG. 2b, divided by the light scattering
image of FIG. 2c, improves visibility and contrast of a pair of
higher emission 1-mm diameter ductal carcinoma lesions 210 as
determined by histopathological (i.e., microscopic tissue disease)
assessment (i.e., hematoxylin-eosin stain). In addition, the ratio
image provides better delineation of the tumor margins. This effect
is also demonstrated in the examples shown in succeeding FIGS. 3
through 5. Furthermore, FIG. 2e shows an inter-image ratio of a
cross-polarized light scattering 1000 nm band-pass image (not
shown) divided by the cross-polarized light scattering 700 nm
band-pass image of FIG. 2c. FIG. 2f shows an inter-image ratio of
cross-polarized light scattering 700 nm band-pass image of FIG. 2c
divided by the autofluorescence image after 532 nm illumination of
FIG. 2a.
[0049] From the images of the specimen shown in FIG. 2, only the
NIR fluorescence image under 632.8-nm excitation FIG. 2b and the
ratio image of FIG. 2d show a correlation with the assessment of
lesions 210. The integrated NIR emission intensity arising from
these cancerous parts of the sample is higher than surrounding
tissue 215 of FIGS. 2b, 2d, and 2f, by a factor of about 1.5 in
FIG. 2b and a factor of about 1.6 in FIG. 2d, FIG. 2e and FIG. 2f
show the presence of adipose tissue 220 with a higher average
intensity of adipose tissue component 220 compared to the
surrounding connective tissue 215 of FIG. 2e and FIG. 2f of about
0.25 and 2.5 for the image shown in FIG. 2e and FIG. 2f,
respectively. The ratio images of FIG. 2d and FIG. 2f reveal high
contrast and increased visibility of different tissue components
(i.e., lesions 210 of FIG. 2d and adipose 220 tissue of FIG. 2f,
respectively) while subjecting specimen 204 to different
illumination wavelengths and by different inter-image operations.
Thus, the present invention can be advantageous in increasing the
visibility of various tissue components in a specimen by varying
system parameters (i.e., illumination wavelength, spectral
band-pass region, inter-image operations, etc.).
[0050] FIGS. 3 shows a set of images obtained from two liver
specimens. FIG. 3a, 3b, and 3c, show a histologic section of a
specimen 206, taken from a benign growth as determined by clinical
assessment. FIGS. 3d, 3e, and 3f, show a histologic section of a
specimen 208 of a well-circumscribed 0.8.times.0.5-cm nodule. The
histologic features of the nodule, as determined by clinical
assessment, are those of a hepatoblastoma (i.e., a tumor of the
liver). FIGS. 3a and 3d are NIR autofluorescence images recorded
under 632-nm illumination, respectively. FIGS. 3b and 3e are NIR
cross-polarized light scattering images recorded under 700-nm
illumination respectively. FIGS. 3c and 3f are the resultant ratio
images of the autofluorescence images FIGS. 3a and 3d over light
scattering images 3b and 3e, respectively. Benign tumor 310 is
visible in specimen 206 of image 3c. The digitized intensity in the
area of the benign tumor 310 is higher between about 0.20 when
compared to that of the adjacent normal liver tissue 315. However,
organ composition, depth of tissue components, and illumination and
inter-image operations, may vary the digitized intensity
information so as to best visualize specific tissue components.
[0051] It is noted however, that cancer nodule 320, in specimen 208
of FIGS. 3d, 3e, and 3f, is shown as an even brighter in intensity
feature with respect to a surrounding normal tissue 325. More
specifically, this intensity difference between tissue components
320, and adjacent normal tissue 325 is further increased in NIR
autofluorescence image FIG. 3d under 632.8-nm excitation by a
factor of about 0.65. In FIG. 3e (cross-polarized light scattering
image under 700-nm illumination) the digitized intensity in the
area of the cancer nodule 320 is higher by a factor of about 0.15
when compared to that of the adjacent normal liver tissue 325.
However, the highest contrast accompanied by best visualization of
tissue margins of cancer nodule 320 is in the ratio image shown in
FIG. 3f, wherein an overall intensity difference between cancer
nodule 320 and adjacent normal tissue 325 is a factor of up to
about 0.90.
[0052] FIGS. 4a, 4b, and 4c, and FIGS. 5a-g show images of the
method of the present invention in delineating cancerous uterine
and bladder tissue from adjacent normal tissue, respectively. FIGS.
4a and 5a show NIR autofluorescence images of the uterine and
bladder organs under 632-nm illumination respectively while FIGS.
4b and 5b show NIR cross-polarized images of the respective organs
under 700-nm illumination. For the uterine specimen, FIG. 4c (i.e.,
the ratio of NIR cross-polarized image under 700-nm illumination of
FIG. 4b over NIR autpofluorescence image under 632-nm illumination
of FIG. 4a), shows a contrast ratio that clearly differentiates
cancerous uterine tissue 410 from surrounding tissue, including
normal tissue 420. Specifically, FIG. 4c shows a contrast ratio of
digitized counts of cancerous tissue 410 from adjacent normal
tissue 420 by a factor of at least 0.65. However, as discussed
herein before, tissue depth, composition, etc., results in
different contrast ratios but with the same qualitative visual
assessment.
[0053] FIG. 5c (i.e., the ratio of NIR cross-polarized image under
700-nm illumination FIG. 5b over NIR autofluorescence image under
632-nm illumination FIG. 5a) shows a high visibility, high contrast
bladder cancerous tissue 510 from surrounding normal bladder tissue
520 similar to the case for uterine tissue shown in FIG. 4. FIG. 5a
shows necrotic tissue 505 (cells that have died as a result of
cancerous growth) as a high intensity region. Moreover, FIG. 5a
(NIR autofluorescence image under 632-nm illumination) and FIG. 5b
(NIR cross-polarized image under 700-nm illumination) also show
cancerous tissue 510 as a dark feature that is about 0.40 less in
digitized intensity with respect to the surrounding lighter
featured normal bladder tissue 520. However, better contrast and
better visibility is still the image shown in FIG. 5c as compared
in FIG. 5a and FIG. 5b separately.
[0054] FIG. 5d shows an example of a near-infrared fluorescence
image under 532-nm illumination with the cancerous tissue 510 and
the normal bladder tissue 520 areas denoted with the associated
arrows. FIG. 5e shows a ratio image following division of the NIR
autofluorescence image obtained under 633-nm excitation as shown in
FIG. 5a over that obtained under 532-nm excitation as shown in FIG.
5d. In this case, cancerous tissue 510 has a digitized intensity
that is higher by a factor of about 0.82 compared to that arising
from normal bladder tissue 520. FIG. 5f shows a ratio image
obtained from division of the 700-nm cross-polarized light
scattering image, as shown in FIG. 5a, by the NIR autofluorescence
image under 532-nm excitation as shown in FIG. 5b, whereby
cancerous tissue 510 has a higher digitized intensity by a factor
of about 1.1 compared to normal bladder tissue 520. Therefore array
of images shown in FIGS. 5a-f clearly indicate that cancerous
tissue 510 can be visualized and tumor margins (e.g., the area
separating normal bladder tissue 520) clearly delineated.
Autofluorescence images can help detect and image cancer that is
located in the superficial tissue layers between about 1 and about
2 mm from the surface. The SPDI technique however can reveal the
presence of different tissue components greater than about 2 mm
provided that there is difference in the light scattering
characteristics between the tissue components, namely cancer and
normal tissue. The ratio image shown in FIG. 5g, (obtained by
division of the cross-polarized light scattering image under
1000-nm illumination over that obtained under 700-nm illumination)
shows very small variations (e.g., less than about 3%) between
normal bladder tissue 520 and cancerous tissue 510. This indicates
that the change in the scattering intensity between normal and
cancer tissues remain relatively unchanged as a function of the
illumination wavelength. However, the absolute changes between
normal and cancer tissue may be somewhat different between
specimens from different patients due to various factors, such as,
but not limited to, differences in fluorescence and scattering
arising from tissues from different patients or tissue specimens
having both normal and cancer components in close proximity.
[0055] The present invention thus combines advantageous methods for
tissue component differentiation. The first method examines
polarized light scattering spectral imaging. The images attained
using elastic light scattering delineate differences in absorption
and scattering between tissue components.
[0056] Another advantageous method of the present invention
involves imaging of various tissue types using the NIR emitted
light under, for example, 632.8-nm and 532-nm excitation. The
images attained using this method are useful for monitoring and/or
imaging endogenous fluorescing agents, such as, porphyrins, which
is useful for the detection of neoplastic (i.e., diseased) tissue
formation. In addition, NIR autofluorescence is further enhanced by
utilizing excitation at different wavelengths. For example the 1-mm
tumor lesions in the breast specimen depicted in FIG. 2c are
visible in the NIR autofluorescence images under 632.8-nm
excitation but not under 532-nm excitation. Therefore, different
illumination wavelengths provided by the present invention can
provide insight into different fluorophores that are indicative of
neoplastic human or animal tissue. Moreover, inter-mage operations
of the present invention, such as, but not limited to, light
scattering over NIR autofluorescence images, has particular utility
for high contrast, high visibility images for tissue component
differentiation.
[0057] Utilization of Cystoscopes
[0058] The most common presenting feature of transitional cell
carcinoma (TCC) of the bladder is hematuria, (i.e., a pathological
condition in which the urine contains blood). Diagnosis of such a
tumor is typically performed by outpatient cystoscopic evaluation
(i.e., interior examination of a patient). A patient is as one
procedure, subsequently scheduled for a day-care surgery requiring
anesthesia, cystoscopic examination, and transurethral resection
(TUR). Following this surgery, the patient is discharged home the
same day, and tissue is sent for histologic examination. Less than
about 75% of such tumors have not invaded the bladder muscle and
are called superficial. Initial therapy consists of a TUR.
[0059] If it is determined that the tumor is superficial and of
low-risk of recurrence and progression, the patient is then
followed with cystoscopic examinations in the office about 4 times
a year for about 2 years, then about 2 times a year for about 2
subsequent years, then about yearly. Low-risk tumors have been
defined as grade I, grade II and stage Ta. If the tumor is regarded
as high-risk for tumor recurrence and progression, i.e., all grade
III tumors, T1 grade II and III multi-focal tumors, concomitant
carcinoma in situ in the bladder (CIS), or tumors that are sessile
(i.e., permanently attached) these patients will receive
intravesical (i.e., within the bladder) therapy as an outpatient.
They will then undergo the same cystoscopic follow-up as outlined
above.
[0060] TCC tumors may recur after resection because: 1) bladder
tumors are multi-focal lesions by nature and subsequent tumors
arise from areas of carcinoma in situ in the bladder (CIS), that
are not yet visible, 2) the area of the tumor is not fully
resected, 3) the urothelium is injured during the resection process
and seeding by TCC cells occurs, and/or 4) recurrence represents a
new tumor development. Therefore, the embodiment detailed
hereinafter provides a benefit in the management of diseases, such
as, TCC, to the patient, doctor, and healthcare system.
[0061] FIG. 6(a) shows an example schematic layout of a high
sensitivity unit (HSU) system, capable of collecting NIR
autofluorescence images with sufficiently low, (i.e., less than
about 1 sec), exposure time, and is generally designated by
reference numeral 200. System 200, includes an illumination
assembly 110 having at least one, often at least two, and more
often, at least six substantially monochromatic CW lasers, 120,
122, 124, 126, 128, 130, such as, but not limited to, diode lasers,
frequency doubled and tripled diode lasers, light emitting diodes
(LEDs), broadband light sources wherein a substantially
monochromatic spectral band is capable of being selected, and/or
gas lasers that may be optically directed, by, for example, one or
more mirrors 132, to a wavelength selection means 134, such as, but
limited to, computer controlled high-speed galvanometer scanner
mirrors, optical switches, and/or manual operation. A selected
monochromatic laser, such as, for example laser 120, by wavelength
selection means 134, is optically coupled to an interior
examination device 136, such as, for example, an endoscope or a
ureterscope, more often a cystoscope, by an external illumination
fiber bundle 138. Illumination from laser 120, is delivered at a
point of interest inside a patient's bladder through an
illumination output 142, as illustrated in FIG. 6(b), of interior
examination device 136. An image, (not shown) is formed by a lens
144, as shown in FIG. 6(b). Such a lens 144 is configured at the
output of interior examination device 136, and is capable of
relaying radiation to project an image on an imaging device 146,
such as, for example, a liquid nitrogen cooled CCD camera, a
two-dimensional array detector, and/or an on-chip amplification CCD
camera, as shown in FIG. 6(a), via coupling optics 148 and after
passing through one or more optical filters 152. The output of
imaging device 146 is capable of being characterized by a means,
such as, but not limited to, a computer 154 configured with image
processing software, e.g., a laptop computer or a personal
computer, and/or a human operator, such as, for example, a
pathologist. The operator evaluates the tissue components after for
example, inter-image operation display by one or more visualization
devices 156, to distinguish tissue features deemed important
information to his or her trained eye.
[0062] In one embodiment, optical filter 152, is arranged to
include a 670-nm long-pass filter that allows transmission for
image acquisition only of the far-red and NIR photons. For
autofluorescence measurements, excitation lasers having a lower
wavelength than an example cutoff wavelength (e.g., 670 nm) of
filter 152, such as, for example, lasers having illumination
wavelengths of 405-nm, 532-nm and/or 632-nm, will therefore pass
only the NIR autofluorescence light through such an example filter
152 while scattered light at the excitation wavelengths, e.g.,
405-nm, 532-nm and/or 632-nm, will be rejected. For the formation
of light scattering images, lasers with longer wavelengths such as,
for example, 670-nm, 820-nm and/or 970-nm, operate at higher
wavelengths than the cut-off wavelength of an example 670-nm
optical filter 152, and as a result, all images will pass through
filter 152.
[0063] An image, can be captured by imaging device 146, via, for
example, an on-chip charge amplification CCD camera, and
characterized by a computer configured with image processing
software and/or an operator after visualization by display means
156. CCD cameras that are based on on-chip charge multiplication
gain technology can have gain factors as high as about 1000.times.,
which allows short exposure times of less than about 1 sec for
autofluorescence images. Such a technology allows camera weights of
about 1 Kgr to be manageable so that an operator is capable of
holding such a camera operatively attached to interior examination
device 136, such as, a cystoscope, in a comfortable manner to allow
such an operator to remain still while imaging device 146 is
capturing an image. Two types of cystoscopes can be adapted into
the present invention and are standard equipment at the UC Davis
Medical Center located in Sacramento, Calif. One such cystoscope is
flexible and is used in the outpatient setting and is based on
image preserving fiber bundle technology. A second cystoscope is
not flexible and is used during transurethral resection. It is
based on image transmission using fiber optic image conduits
technology. Each capable cystoscope has an image collection element
(i.e., an image preserving fiber bundle or glass rod) and a means
to illuminate a sample (e.g., bladder). Illumination is delivered
using a fiber bundle that is part of the assembly of the
cystoscope. This internal illumination fiber is coupled to an
external light source through external illumination fiber bundle
138 that attaches at an input of a cystoscope's internal fiber
bundle.
[0064] As another example arrangement, when implementing SPDI
subsurface imaging, illumination output 142 is arranged with a
polarization filter (not shown) either fixedly attached or coupled
with a fixture (not shown) capable of being removed for specific
applications. A polarization filter (not shown), having a
polarization orientation orthogonal with respect to the
polarization filter at output 142, is also arranged with coupling
optics 148. By such an arrangement, an image is formed by photons
that have polarization orientation orthogonal to that of the
illumination. Such formed images are then capable of being
characterizing by, for example, a computer 154 configured with
image processing software and/or an operator after
visualization.
[0065] As a further arrangement, system 200 is capable of being
adapted to operate as a "maximum speed unit" (MSU) system. Such a
system 200, provides simultaneous images of a tissue sample (not
shown) from at least two, and more often, at least three
illumination wavelengths, e.g., 670-nm, 820-nm and, 970-nm. In this
embodiment, a second illumination assembly (not shown) is
configured to direct one or more wavelengths to tissue components
of interest through optically coupled interior examination device
136. Lens 144, as shown in FIG. 6(b), can optically relay
simultaneous images from the illuminated tissue components for
capture by imaging device 146. In addition, lens 144 of interior
examination device 136, such as a cystoscope, is capable of being
optically coupled to an image preserving fiber bundle (not shown)
to also relay simultaneous images to coupling optics 148 for
capture by imaging device 146. These spectral image components are
spatially separated, such as, for example, by using appropriate
spectral dispersion optics (not shown), and/or mirrors before
projection onto imaging device 146. In this arrangement, imaging
device 146 can include a two-dimensional detector (e.g., a
400.times.1300 pixels CCD) so that each spectral image component is
capable of being simultaneously projected on different parts of
imaging device 146. Appropriate software implemented by computer
154 can then perform inter-image operations between image
components associated with different illumination wavelengths for
characterizing the illuminated tissue components in accordance with
the methodology of the SPDI technique.
[0066] As an example protocol, an examination of a tissue region
may involve acquisition or generation of up to about ten images
such as, three cross polarized images under 670-nm (670 CPI),
820-nm (820 CPI) and, 970-nm (970 CPI) illumination, three SDPI
images from subtraction following normalization of the cross
polarized images under illumination at 970-nm and 670-nm (e.g., 9-7
SPDI), 970-nm and 820-nm (e.g., 9-8 SPDI), 820-nm and 670-nm (e.g.,
8-7 SPDI), two NIR autofluorescence images under 532-nm (532 AFI)
and 632.8-nm (633 AFI) excitation, an autofluorescence ratio image
of 633-nm illumination over 532-nm excitation (AFRI) and a ratio
image of light scattering at 700-nm over the autofluorescence image
under 532-nm excitation (SARI). An operator evaluates the generated
images with four example scenarios shown in Table 1:
1TABLE 1 Feature is visible on image: 670 820 970 9-7 9-8 8-7 532
633 CPI CPI CPI SPDI SPDI SPDI AFI AFI AFRI SARI Evaluation yes yes
yes yes no yes yes yes yes yes Superficial cancer no no no no yes
yes no no no no Subsurface tumor yes yes yes yes yes yes yes yes
yes yes Cancer extends from surface to muscle yes yes yes yes no
yes yes yes no yes Surface lesion, not cancer
[0067] During operation of the system, the operator may start by
displaying images in a monitor that will help determine if there is
cancer on the surface (i.e. superficial cancer as shown in the last
column). If there is no surface cancer, the operator may search for
subsurface tumors (i.e., SPDI images). If there is cancer on the
surface, the operator may use the SPDI images to evaluate the depth
of penetration of the tumor (i.e., the cancer extends from the
surface to muscle or the identified tissue is a surface lesion and
not cancerous).
[0068] Embodiments of system 200, as shown in FIG. 6a are
beneficial in multiple ways. As some examples, system 200 enhances
the follow-up of patients with Ta and T1 tumors, it increases the
accuracy of detecting recurrent tumors, Ta lesions could be treated
in the office rather than the operating room, and pink areas which
can be confused as either CIS or inflammation following
intravesical therapy could be accurately analyzed. In addition,
system 200 is capable of being implemented on ureterscopes to
diagnose lesions of the ureter and renal pelvis. Finally, there
would be a small group of patients in whom system 200 could be
adapted for percutaneous, (i.e., through the skin), access to the
upper tract.
[0069] Accordingly, system 200 provides an enormous benefit to
patients, doctors, and the healthcare system in managing and
treating disease such as cancer (e.g., TCC).
[0070] It should be understood that the invention is not intended
to be limited to the particular forms disclosed. Rather, the
invention is to cover all modifications, equivalents, and
alternatives falling within the spirit and scope of the invention
as defined by the following appended claims.
* * * * *